Radiopharmaceuticals are drugs containing a radionuclide, and are used routinely in nuclear medicine department for the diagnosis or therapy of various diseases. They are mostly small organic or inorganic compounds with definite composition. They can also be macromolecules such as antibodies and antibody fragments that are not stoichiometrically labeled with a radionuclide. Radiopharmaceuticals form the chemical basis for nuclear medicine, a group of techniques used for diagnosis and therapy of various diseases. The in vivo diagnostic information is obtained by intravenous injection of the radiopharmaceutical and determining its biodistribution using a gamma camera. The biodistribution of the radiopharmaceutical depends on the physical and chemical properties of the radiopharmaceutical and can be used to obtain information about the presence, progression, and the state of disease.
Radiopharmaceuticals can be divided into two primary classes: those whose biodistribution is determined exclusively by their chemical and physical properties; and those whose ultimate distribution is determined by their receptor binding or other biological interactions. The latter class is often called target-specific radiopharmaceuticals.
In general, a target specific radiopharmaceutical can be divided into four parts: a targeting molecule, a linker, a Bifunctional Chelator (BFC), and a radionuclide. The targeting molecule serves as a vehicle which carries the radionuclide to the receptor site at the diseased tissue. The targeting molecules can be macromolecules such as antibodies. They can also be small biomolecules (BM): peptides, peptidomimetics, and non-peptide receptor ligands. The choice of biomolecule depends upon the targeted disease or disease state. The radionuclide is the radiation source. The selection of radionuclide depends on the intended medical use (diagnostic or therapeutic) of the radiopharmaceutical. Between the targeting molecule and the radionuclide is the BFC, which binds strongly to the metal ion via several coordination bonds and is covalently attached to the targeting molecule either directly or through a linker. Selection of a BFC is largely determined by the nature and oxidation state of the metallic radionuclide. The linker can be a simple hydrocarbon chain or a long poly(ethylene glycol) (PEG), which is often used for modification of pharmacokinetics. Sometimes, a metabolizeable linker is used to increase the blood clearance and to reduce the background activity, thereby improving the target-to-background ratio.
The use of metallic radionuclides offers many opportunities for designing new radiopharmaceuticals by modifying the coordination environment around the metal with a variety of chelators. The coordination chemistry of the metallic radionuclide will determine the geometry of the metal chelate and the solution stability of the radiopharmaceutical. Different metallic radionuclides have different coordination chemistries, and require BFCs with different donor atoms and ligand frameworks. For "metal essential" radiopharmaceuticals, the biodistribution is exclusively determined by the physical properties of the metal chelate. For target-specific radiopharmaceuticals, the "metal tag" is not totally innocent because the target uptake and biodistribution will be affected by the metal chelate, the linker, and the targeting biomolecule. This is especially true for radiopharmaceuticals based on small molecules such as peptides due to the fact that in many cases the metal chelate contributes greatly to the overall size and molecular weight. Therefore, the design and selection of the BFC is very important for the development of a new radiopharmaceutical.
A BFC can be divided into three parts: a binding unit, a conjugation group, and a spacer (if necessary). An ideal BFC is that which is able to form a stable .sup.99m Tc complex in high yield at very low concentration of the BFC-BM conjugate. There are several requirements for an ideal BFC. First, the binding unit can selectively stabilize an intermediate or lower oxidation state of Tc so that the .sup.99m Tc complex is not subject to redox reactions; oxidation state changes are often accompanied by transchelation of .sup.99m Tc from a .sup.99m Tc-BFC-BM complex to the native chelating ligands in biological systems. Secondly, the BFC forms a .sup.99m Tc complex which has thermodynamic stability and kinetic inertness with respect to dissociation. Thirdly, the BFC forms a .sup.99m Tc complex with a minimum number of isomers since different isomeric forms of the .sup.99m Tc-chelate may have significant impact on the biological characteristics of the .sup.99m Tc-BFC-BM complex. Finally, the conjugation group can be easily attached to the biomolecule.
In simple technetium complex radiopharmaceuticals such as .sup.99m Tc-sestamibi, [.sup.99m Tc(MIBI).sub.6 ].sup.+ (MIBI=2-methoxy-2-methylpropyl-isonitrile) and .sup.99m Tc-bicisate, [.sup.99m TcO (ECD)] (ECD=1,1-ethylene dicysteine diethyl ester), the ligand (MIBI or ECD) is always present in large excess. The main factor influencing the .sup.99m Tc-labeling kinetics is the nature of the donor atoms and the radiolabeling conditions. For receptor-based target specific radiopharmaceuticals, however, the use of large amount of BFCA-BM may result in receptor site saturation, blocking the docking of the .sup.99m Tc-labeled BFC-BM, as well as unwanted side effects. In order to avoid these problems, the concentration of the BFC-BM in the radipharmaceutical kit has to be very low (10.sup.-6 -10.sup.-5 M). Otherwise, a post-labeling purification is often needed to remove excess unlabeled BFC-BM, which is time consuming and thus not amenable for clinical use. Compared to the total technetium concentration (.about.5.times.10.sup.-7 M) in 100 mCi of [.sup.99m Tc]pertechnetate (24 h prior-elution), the BFC-BM is not in overwhelmingly excess. Therefore, the BFC attached to the biomolecule must have very high radiolabeling efficiency in order to achieve high specific activity, the amount of unlabeled BFC-BM conjugate used to synthesize the radiopharmaceutical. Various BFCs have been used for the .sup.99m Tc-labeling of biomolecules, and have been extensively reviewed (Hom, R. K. and Katzenellenbogen, J. A. Nucl. Med. Biol. 1997, 24, 485; Dewanjee, M. K. Semin. Nucl. Med. 1990, 20, 5; Jurisson, et al Chem. Rev. 1993, 93, 1137; Dilworth, J. R. and Parrott, S. J. Chem. Soc. Rev. 1998, 27, 43; Liu, et al Bioconj. Chem. 1997, 8, 621; Liu, et al Pure & Appl. Chem. 1991, 63, 427; Griffiths, et al Bioconj. Chem. 1992, 3, 91).
The use of hydrazines and hydrazides as BFCs to modify proteins for labeling with radionuclides has been recently disclosed in Schwartz et al U.S. Pat. No. 5,206,370. For labeling with technetium-99m, the hydrazino-modified protein is reacted with a reduced technetium species, formed by reacting pertechnetate with a reducing agent in the presence of a chelating dioxygen ligand. The technetium is bonded through what are believed to be hydrazino or diazenido linkages with the coordination sphere completed by the coligands such as glucoheptonate and lactate. Bridger et al European Patent Application No. 93302712.0 discloses a series of functionalized aminocarboxylates and their use for the radiolabeling of hydrazino-modified proteins. The improvements are manifested by shorter reaction times and higher specific activities for the radiolabeled protein. The best example is tricine.
Archer et al, European Patent application 90914225.9 discloses a series of technetium-99m complexes having a ternary ligand system comprised of a hydrazino or diazenido ligand, a phosphine ligand and a halide, in which the substituents on the hydrazido or diazenido ligand and those phosphine ligand can be independently varied. This disclosure does not teach or suggest how to achieve the superior control of biological properties that will result from a ternary ligand system in which the substituents on the three types of ligands can be independently varied. In addition, the radiopharmaceuticals described by Archer et al are formed in low specific activity. Therefore, there remains a need for new ternary ligand systems which form radiopharmaceuticals with high specific activity.
In WO 97/33627 the synthesis of novel radiolabeled platelet glycoprotein IIb/IIIa receptor antagonists as imaging agents for thromboembolic disorders is disclosed. Hydrazinonicotinamide (HYNIC) is used as the BFC for the modification of cyclic compounds while an aminocarboxylate such as tricine and an imine-N containing heterocycle are coligands. The combination of HYNIC-BM, tricine and a monodentate imine-N containing heterocycle produces a unique and versatile ternary ligand system that forms ternary ligand technetium complexes [.sup.99m Tc(HYNIC-BM)(tricine)(heterocycle)] with high solution stability and only two detectable isomeric forms (due to chiral substituents on HYNIC).
The coligand has profound impact on the hydrophilicity and biological properties of the ternary ligand technetium complex [.sup.99m Tc(HYNIC-BM) (tricine)(heterocycle)]. Thus, it is desirable to discover new coligands.